TCS is a plant protein which is obtained from the Trichosanthes kirilowii root tuber. The protein, which is also known as alpha-TCS (Law) and Radix trichosanthis (Kuo-Fen), is a basic, single-chain protein which has been reported to containing about 224 (Gu) to 234 (Xuejan) amino acid residues, and reported to have a molecular weight of about 24,000 daltons. The protein sequence of TCS has been completed (Gu; Wang), and a molecular model has been derived from cytofluorographic X-ray analysis (Kezhan).
It has been shown that TCS is a potent inhibitor of protein synthesis in a cell-free lysate system (Maraganove). This activity is consistent with the observed homology in amino acid sequence between TCS and the A chain of ricin, a ribosome-inactivating protein (RIP) which shows amino acid homology with a number of other RIPs, including abrin A chain (Olnes, 1982, 1987) and modeccin (Olnes, 1982), and various single-chain ribosome-inactivating proteins, such as pokeweed anti-viral protein (PAP) (Irvin), RIPs from a variety of other plants (Coleman; Grasso; Gasperi-Campani) and the A subunit of Shiga-like toxins from E. coli (Calderwood).
TCS, or plant extracts containing TCS, have been used in China as an abortifacient agent for inducing abortion in humans, particularly during midtrimester (14 to 26 weeks). As such, the drug has been administered by intramuscular, intravenous, or intraamniotic routes. The phenomenon of mid-term abortion has been attributed to the selective destruction of placental villi. Other studies indicate that the syncytiotrophoblast is preferentially affected (Hsu; Kao) and that secretion of hCG may be impaired (Xiong). TCS has also been shown to have a suppressive effect on human choriocarcinoma, and the protein appears to be able to pass the blood/brain barrier (Hwang).
Co-owned U.S. Pat. No. 4,795,739 for "Method of Inhibiting HIV" describes the use of TCS for inhibiting HIV (Human Immunodeficiency Virus) proliferation in infected human T cells and macrophages, for treating HIV infection in humans. In view of the significant therapeutic use of TCS, it would be valuable to obtain the protein in substantially pure form by preparation methods which are suited to large-scale processing.
Methods of preparing TCS have been reported (Yeung). These methods typically involve an initial aqueous extraction of fresh root tubers of T. kirilowii, and lyophilization of the extract supernatant. The resulting powder is dissolved in water medium, followed by addition of solvent, such as an aqueous acetone mixture, to yield a precipitate containing TCS. After removal of the precipitate by centrifugation, additional solvent is added to the supernatant, yielding a second TCS-containing precipitate fraction on centrifugation. Both precipitates are re-extracted with aqueous medium, giving soluble protein fractions containing partially purified TCS.
The soluble fraction from the second-solvent precipitate can be further purified by cation exchange column chromatography, using a NaCl gradient to release the protein. The eluate profile from the column indicates the presence of contaminating proteins on both sides of the protein peak identified as TCS.
Alternatively, the soluble fraction from the first-or from the second-solvent precipitate can be purified by an initial pass through an anion exchange resin column, and the eluate further purified by cation exchange column chromatography. Although the resulting protein is somewhat more pure than that from the method involving cation exchange chromatography alone, the cation exchange elution profile still shows the presence of prominent side peaks, as evidenced by size-exclusion HPLC.
Studies carried out in support of the present application, and discussed below, indicate that the TCS protein isolated by the above methods contains readily detectable contaminating proteins. Based on the observed hemagglutination activity of the purified protein preparation, at least one of the contaminants is a hemagglutinin. The above-described purification methods are also difficult to adapt to large scale, in view of the need for volatile organic solvent extraction.